1. Massive antibody discovery used to probe structure-function relationships of the essential outer membrane protein LptD.
- Author
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Storek KM, Chan J, Vij R, Chiang N, Lin Z, Bevers J 3rd, Koth CM, Vernes JM, Meng YG, Yin J, Wallweber H, Dalmas O, Shriver S, Tam C, Schneider K, Seshasayee D, Nakamura G, Smith PA, Payandeh J, Koerber JT, Comps-Agrar L, and Rutherford ST
- Subjects
- Animals, Anti-Bacterial Agents pharmacology, Binding Sites, Epitope Mapping, Epitopes metabolism, Escherichia coli growth & development, Escherichia coli metabolism, Mice, Inbred BALB C, Protein Structure, Secondary, Rats, Sprague-Dawley, Structure-Activity Relationship, Antibodies, Monoclonal metabolism, Bacterial Outer Membrane Proteins chemistry, Bacterial Outer Membrane Proteins metabolism, Escherichia coli Proteins chemistry, Escherichia coli Proteins metabolism
- Abstract
Outer membrane proteins (OMPs) in Gram-negative bacteria dictate permeability of metabolites, antibiotics, and toxins. Elucidating the structure-function relationships governing OMPs within native membrane environments remains challenging. We constructed a diverse library of >3000 monoclonal antibodies to assess the roles of extracellular loops (ECLs) in LptD, an essential OMP that inserts lipopolysaccharide into the outer membrane of Escherichia coli . Epitope binning and mapping experiments with LptD-loop-deletion mutants demonstrated that 7 of the 13 ECLs are targeted by antibodies. Only ECLs inaccessible to antibodies were required for the structure or function of LptD. Our results suggest that antibody-accessible loops evolved to protect key extracellular regions of LptD, but are themselves dispensable. Supporting this hypothesis, no α-LptD antibody interfered with essential functions of LptD. Our experimental workflow enables structure-function studies of OMPs in native cellular environments, provides unexpected insight into LptD, and presents a method to assess the therapeutic potential of antibody targeting., Competing Interests: KS, JC, RV, NC, ZL, JB, CK, JV, YM, JY, HW, OD, SS, CT, KS, DS, GN, PS, JP, JK, LC, SR Employee of Genentech, Inc, a member of the Roche Group, and shareholder in Roche., (© 2019, Storek et al.)
- Published
- 2019
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